Using murine models, we have shown that osteoclast-derived factors promote the recruitment and differentiation of osteoblast lineage cells. Because osteoclasts release and activate TGF-? from the bone matrix and are TGF-??responsive cells, we asked whether osteoclast TGF-? responses contribute to the coupling of bone resorption to subsequent bone formation. In mice, expression of a dominant negative TGF-? receptor in osteoclasts (Tgfbr2OclKO) caused marked osteopenia in the absence of any impact on osteoclast numbers. Importantly, osteoblast numbers and bone formation were reduced, supporting that osteoclast TGF-? responses contribute to osteoblast recruitment and/or differentiation. Wnt1 is a crucial regulator of normal bone formation. We found that TGF-? treatment of wildtype osteoclasts or culturing osteoclasts on bone resulted in marked increases in Wnt1 mRNA and protein. This response was blunted in Tgfbr2OclKO osteoclasts. Interestingly, osteoclast Wnt1 protein expression was much lower in Tgfbr2OclKO bones, suggesting that Wnt1 is a candidate TGF-?-induced osteoclast coupling factor. This leads to our central hypothesis that locally released TGF-? acts on osteoclasts to produce coupling factors including Wnt1, which stimulate bone formation by enhancing osteoblast differentiation. We have designed studies to test this hypothesis in murine models (Aim 1) and human subjects (Aim 2). Our Specific Aims to test our hypothesis are: 1. In murine models, investigate the impacts of loss of functional osteoclast TGF-? receptor signaling on osteoblasts and the roles of osteoclast lineage Wnt1 in bone metabolism. We will (A) Evaluate Tgfbr2OclKO mouse osteoclast lineage cell coupling factor expression and osteoblast lineage cell responses in the altered Tgfbr2OclKO microenvironment, (B) Determine the role of osteoclast Wnt1 osteoclast production in osteoclast-osteoblast coupling and (C) Define the mechanism by which matrix bound TGF-? regulates coupling factor production and responses by osteoblasts. 2. Determine the effect of osteoclast reduction on coupling factor production and osteoblast progenitors in vivo in postmenopausal women. We will (A) Examine levels of potential coupling factors in the bone microenvironment and (B) Evaluate osteoclast lineage cell coupling factor gene expression and their target gene expression levels in osteoblasts. We expect that these studies will reveal roles for osteoclast-derived TGF-?-induced coupling factors, including Wnt1, in bone formation and turnover. In order to move the concept of coupling towards a viable therapeutic target for further development, documentation of its importance in humans is required. This is the goal of Aim 2. The innovative aspects of this project are: (1) the mechanisms by which osteoclasts modulate osteoblasts will be examined in both mice and humans, (2) the role of osteoclast-derived Wnt1 in skeletal development and osteoclast-osteoblast coupling will be determined, and (3) a new approach to evaluate gene expression in osteoblast/osteocyte/osteoclast lineage cells in vivo without in vitro manipulation will be employed.